Protein Concentration Calculator (Bradford, Lowry, BCA)

Accurate protein quantification is essential for biochemical and molecular biology research. Protein concentration calculators convert absorbance data into precise protein amounts.

This article explores three widely used protein assays—Bradford, Lowry, and BCA—detailing formulas, tables, and real-world applications. Learn how to calculate protein concentrations confidently.

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Example Numeric Prompts for Protein Concentration Calculator

• Calculate protein concentration from Bradford assay absorbance at 595 nm = 0.45
• Determine protein concentration using Lowry method with absorbance at 750 nm = 0.32
• Find protein concentration from BCA assay with absorbance at 562 nm = 0.60
• Convert absorbance 0.50 (Bradford) to mg/mL protein concentration using BSA standard curve

Comprehensive Tables of Protein Concentration Values for Bradford, Lowry, and BCA Assays

Protein concentration assays rely on standard curves generated from known protein standards, typically Bovine Serum Albumin (BSA). Below are extensive tables showing typical absorbance values and corresponding protein concentrations for each assay.

Fundamental Formulas for Protein Concentration Calculation

Protein concentration is typically calculated by interpolating the absorbance of an unknown sample against a standard curve generated from known protein concentrations. The general formula is:

Where:

• Absorbance: Measured optical density of the unknown sample at the assay-specific wavelength.
• Intercept: Y-intercept of the standard curve linear regression.
• Slope: Slope of the standard curve linear regression.

Bradford Assay Formula

The Bradford assay uses Coomassie Brilliant Blue dye binding to proteins, measured at 595 nm. The standard curve is often linear within 0.1 to 1.5 mg/mL protein range.

• C: Protein concentration (mg/mL)
• A₅₉₅: Absorbance at 595 nm
• b: Y-intercept of Bradford standard curve
• m: Slope of Bradford standard curve

Typical values for BSA standard curve:

• Slope (m): ~1.1 absorbance units per mg/mL
• Intercept (b): ~0.0 to 0.05 absorbance units

Lowry Assay Formula

The Lowry assay is based on the reduction of Folin-Ciocalteu reagent by protein-copper complexes, measured at 750 nm. It is sensitive but more time-consuming.

• C: Protein concentration (mg/mL)
• A₇₅₀: Absorbance at 750 nm
• b: Y-intercept of Lowry standard curve
• m: Slope of Lowry standard curve

Typical BSA standard curve values:

• Slope (m): ~1.9 absorbance units per mg/mL
• Intercept (b): ~0.0 absorbance units

BCA Assay Formula

The Bicinchoninic Acid (BCA) assay relies on the reduction of Cu²⁺ to Cu¹⁺ by proteins and subsequent chelation by BCA, measured at 562 nm. It is highly sensitive and compatible with detergents.

• C: Protein concentration (mg/mL)
• A₅₆₂: Absorbance at 562 nm
• b: Y-intercept of BCA standard curve
• m: Slope of BCA standard curve

Typical BSA standard curve values:

• Slope (m): ~1.6 absorbance units per mg/mL
• Intercept (b): ~0.0 absorbance units

Detailed Real-World Examples of Protein Concentration Calculation

Example 1: Bradford Assay Protein Concentration Calculation

A researcher measures the absorbance of an unknown protein sample at 595 nm and obtains a value of 0.45. The BSA standard curve linear regression equation is:

Where C is protein concentration in mg/mL.

Step 1: Rearrange the equation to solve for C:

Step 2: Substitute the absorbance value:

Result: The protein concentration of the unknown sample is approximately 0.364 mg/mL.

Example 2: BCA Assay Protein Concentration Calculation

In a BCA assay, the absorbance of an unknown sample at 562 nm is measured as 0.80. The BSA standard curve equation is:

Step 1: Rearrange to solve for C:

Step 2: Substitute the absorbance value:

Result: The protein concentration is approximately 0.488 mg/mL.

Additional Technical Considerations for Protein Concentration Calculations

• Standard Curve Preparation: Always prepare fresh standard curves with the same batch of reagents and under identical conditions to ensure accuracy.
• Linear Range: Each assay has a specific linear range; samples with absorbance outside this range should be diluted appropriately.
• Interferences: Detergents, reducing agents, and buffer components can interfere with assays differently. BCA is more tolerant to detergents than Bradford or Lowry.
• Sample Blank: Always run a blank with the sample buffer to subtract background absorbance.
• Replicates: Perform measurements in triplicate to minimize experimental error.
• Assay Sensitivity: Bradford is rapid and simple but less sensitive to low protein concentrations compared to Lowry and BCA.

Summary of Assay Characteristics

References and Further Reading

• Sigma-Aldrich: Bradford Protein Assay
• Thermo Fisher Scientific: BCA Protein Assay Kit
• Lowry et al., 1951 – Protein Measurement with the Folin Phenol Reagent
• Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.

By mastering these protein concentration calculation methods, researchers can ensure precise quantification critical for downstream applications such as enzyme kinetics, protein purification, and structural studies.